Trachoma is a chlamydial disease of the developing world whereas STI are endemic to both developed and undeveloped nations. There is currently no licensed vaccine to control these infections. The major challenges to chlamydial vaccine development are; (i) the diseases are caused by multiple chlamydial serovars and protective immunity is serovar specific, (ii) an effective vaccines must target local mucosal immunity of the eye or genital tract, and (iii) effective protective immunity must consist of both antibody and cell mediated immunity to multiple antigens. These requirements have been difficult, if not impossible, to achieve using conventional subunit vaccine approaches. We hypothesized that a live-attenuated vaccine might be the best approach to satisfy these requirements and, in fact, recently described a plasmid-deficient live attenuated trachoma vaccine that protected non-human primates against experimental trachoma (J. Exp. Med 2011, 208:2217). We are currently pursuing an investigational new drug for the vaccine with the FDA, and if approved, will initiate phase I clinical trials in the near future. Unfortunately, there is little commercial market for a trachoma vaccine. However, there is a substantial commercial market for a chlamydial STI vaccine. Generating a live-attenuated vaccine for chlamydial STI is more challenging than our trachoma vaccine because of the multiple (8) chlamydial serovariants that can cause urogenital infection. In concept, however, this goal is achievable based on current knowledge of the successful trachoma vaccine. We know that plasmid gene(s) are attenuating virulence factors for the trachoma vaccine and we have now identified a single plasmid gene, plasmid protein gene 4 (ppg4) that is the primary virulence determinant. It is now feasible to use this information to construct a single STI strain with a genetically modified plasmid lacking ppg4 (bad gene) as a shuttle vector to express multiple chlamydial protective antigens (good genes). This will be achieved by completing the following specific aims; (i) delete ppg4 from the plasmid and show the strain harboring the ppg4 mutant is fully attenuated in animal models of infection. To date, we have deleted ppg4 and are currently evaluating the mutant for attenuation in animal models; (ii) clone multiple chlamydial ompA genes (ompA is the major chlamydial serotyping and protective antigen) into the genetically attenuated ppg4 deficient plasmid and use the plasmid as a shuttle vector to express multiple ompA genes in a single strain capable of inducing broad protective coverage against multiple chlamydial STI serotypes. To date, we have successfully expressed several ompA genes using the attenuated plasmid vector in single chlamydial strain; (iii) further genetically engineer the OmpA multivalent chlamydial attenuated STI vaccine to express heterologous protective antigens of other medically important viral STI pathogens such as HIV, HPV, and Herpes virus at the genital mucosae. A second approach to achieve this goal will be to use the attenuated plasmid as shuttle vector to clone multiple chlamydial ompA protective genes, or heterologous pathogen genes, into the chlamydial genome using the well characterized non-essential tryptophan operon as the recombination locus. Thus, this technology has the attractive potential to generate a single live-attenuated chlamydial vaccine strain capable of protecting against both chlamydial STI and other STD pathogens.